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    黄精调控PINK1介导的线粒体自噬延缓大鼠内皮祖细胞复制性衰老

    Polygonati Rhizoma Attenuate the Replicative Senescence of Endothelial Progenitor Cells of Rats Via PINK1 Mediated Mitophagy

      摘要
    • 摘要:
      目的  动态观察黄精含药血清对大鼠骨髓内皮祖细胞(endothelial progenitor cells，EPCs)复制性衰老进程中线粒体功能和线粒体自噬水平的影响，探讨黄精能否通过调节磷酸酶和张力蛋白同源物诱导激酶1(PTEN-induced putative kinase 1, PINK1)介导的线粒体自噬延缓EPCs衰老。
      方法 分离培养大鼠骨髓EPCs并鉴定，继续将EPCs培养至第4、6、8代，每代各分为7组：对照组(10%空白血清)，黄精低、中、高剂量组(10%黄精低、中、高剂量含药血清)，黄精低、中、高剂量+3-甲基腺嘌呤(3-methyl adenine，3-MA)组。各组干预48 h后，采用β-半乳糖苷酶染色法检测细胞衰老程度；CCK-8 法检测细胞增殖功能；Transwell小室检测细胞迁移功能；体外成血管试剂盒检测细胞成小管功能；JC-1法检测细胞线粒体膜电位；化学发光法检测胞内ATP含量；流式细胞仪检测胞内ROS水平；Western blotting检测线粒体自噬相关蛋白PINK1、帕金蛋白(Parkin)、微管相关蛋白轻链3(microtubule-associated protein light chain 3，LC3)、P62的表达。
      结果 EPCs在体外传代培养至第4、6、8代，其衰老程度逐渐加重，细胞增殖、迁移和成小管功能损伤逐渐加重；胞内ROS水平逐渐升高，线粒体膜电位和ATP 含量逐渐降低；PINK1、Parkin、LC3蛋白表达下调，P62蛋白上调。经黄精干预后，EPCs衰老程度减轻，细胞增殖、迁移和成小管功能有所改善；胞内ROS水平降低，线粒体膜电位和ATP 含量升高；PINK1、Parkin、LC3蛋白表达上调，P62蛋白表达下调；3-MA可显著抑制黄精对EPCs的上述作用。
      结论  黄精可通过提升PINK1介导的线粒体自噬水平，改善线粒体功能，从而延缓大鼠EPCs的复制性衰老。

       

      Abstract:
      OBJECTIVE  To observe dynamically the effect of Polygonati Rhizoma(PR) on the mitochondrial function and mitophagy in the replicative senescence process of endothelial progenitor cells(EPCs) derived from rat bone marrow, investigating the attenuate effect of PR on the senescence of EPCs is related with PTEN-induced putative kinase1(PINK1) mediated mitophagy.
      METHODS  EPCs derived from rat bone marrow were cultured and characterized. The 4th, 6th, and 8th passages of EPCs were divided into 7 groups: control group(treated with 10% blank serum), PR low, middle and high dose group(treated with 10% PR low, medium, and high dose drug-containing serum, respectively), PR low, middle and high dose groups with 3-methyl adenine(3-MA). Following a 48h intervention, the positive rate of cell senescence was assessed using β-galactosidase. Cellular function of proliferation, migration and tubule formation were evaluated using CCK-8, transwell chamber, and in vivo angiogenesis kit, respectively. Additionally, the levels of reactive oxygen species(ROS), mitochondrial membrane potential(MMP), and ATP content were measured using flow cytometry, JC-1 and chemiluminescence method. The expression of mitophagy related proteins PINK1, Parkin, microtubule-associated protein light chain 3(LC3) and P62 were detected by Western blotting.
      RESULTS  EPCs were subcultured to the 4th, 6th and 8th passage, the positive rate of cell senescence increased gradually, accompanied by a significant decrease in proliferation, migration and tubule formation of EPCs. The level of ROS increased but the MMP and ATP content both decreased. The expression of PINK1, Parkin, and LC3 were down-regulated while P62 protein was up-regulated. However, PR treatment could reduce the positive rate of cell senescence, improve the cell proliferation, migration and tubular function, decrease the ROS level and increase the MMP and ATP content. The expression of PINK1, Parkin and LC3 were up-regulated while P62 protein was down-regulated after RP treatment. 3-MA significantly inhibited the above effects of RP on EPCs.
      CONCLUSION PR could attenuate the replicative senescence of EPCs in rats by improving the mitochondrial function through PINK-mediated mitophagy.

       

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